Detection of the Enzymatic Cleavage of DNA through Supramolecular Chiral Induction to a Cationic Polythiophene

Abstract

Water-soluble π-conjugated polymers are increasingly envisioned in biosensors, in which their unique optical and electronic properties permit a highly sensitive detection of biomolecular targets. In particular, cationic π-conjugated polymers are attractive for DNA sensing technologies, through the use of the fluorescence signals either in physiological solutions or in thin films. However, in the context of enzymatic activity assays, fluorescence-based methods require covalently labeling DNA with a dye or an antibody and are limited to short time scale due to dye photobleaching. In this frame, we report here a novel possible approach to probe the cleavage of DNA by a restriction enzyme, in continuous and without covalently labeled DNA substrate. This is achieved by exploiting unique chiroptical signals arising from the chiral induction of DNA to a poly­[3-(6′-(trimethylphosphonium)­hexyl)­thiophene-2,5-diyl] upon interaction. The cleavage of DNA by HpaI, an endonuclease enzyme, is monitored through circular dichroism (CD) signals in the spectral range where the polymer absorbs light, i.e., far away from the spectral ranges of both DNA and the enzyme. We compare the results to a conventional noncontinuous assay by polyacrylamide gel electrophoresis, and we demonstrate that induced CD signals are effective in probing the enzymatic activity. By means of molecular dynamics simulations and calculations of CD spectra, we bring molecular insights into the structure of DNA/polymer supramolecular complexes before and after the cleavage of DNA. We show that the cleavage of DNA modifies the dynamics and the organization of the polymer backbone induced by the DNA helix. Altogether, our results provide detailed spectroscopic and structural insights into the enzymatic cleavage of DNA in interaction with a π-conjugated polymer, which could be helpful for developing chiroptical detection tools to monitor the catalytic activity in real time.

Binding Mode Multiplicity and Multiscale Chirality in the Supramolecular Assembly of DNA and a π‐Conjugated Polymer

“…Previously, we showed that the P3HTÀ PMe 3 binding to DNA induces a rearrangement in the distribution of the dihedral angles between thiophene units, from a slight majority of anti configurations between thiophene units (for the pure polymer) to a majority of syn configurations when it interacts with DNA. [14] The change toward a syn P3HTÀ PMe 3 backbone is also observed in each replica of this study. Interestingly, the ratio of syn-to anti-dihedral angles is very similar between the four replicas ( Table 1).…”

“…In a previous study, we proposed a stable binding mode of P3HTÀ PMe 3 around a double-stranded DNA, with preferential adsorption of the thiophene units along the DNA minor-groove, and with the lateral phosphonium groups complexed by the DNA phosphodiester groups. [14] To uncover other stable binding modes, we performed four new MD replicas (named R1 to R4), from 2 μs to 10 μs, see the Methods Section. We follow the time-evolution of the distance between the centers of mass (COM) of both partners (Figure 1), as an average indicator of DNA/polymer proximity within the complexes.…”

“…[13] Such polyplexes were used to monitor in real-time the cleavage of DNA by HpaI (Haemophilus parainfluenzae), an endonuclease enzyme, via chiroptical signals. [14] Thanks to the tremendous progress of computer performance, the accessible size and timescale for performing more 'realistic' simulations of biomolecular systems have increased. [15] In our abovementioned study, [14] experimental ICD results were complemented by MD simulations to bring atomistic insights into the organization and the dynamics of P3HTÀ PMe 3 before and after the cleavage of DNA.…”

“…[14] Thanks to the tremendous progress of computer performance, the accessible size and timescale for performing more 'realistic' simulations of biomolecular systems have increased. [15] In our abovementioned study, [14] experimental ICD results were complemented by MD simulations to bring atomistic insights into the organization and the dynamics of P3HTÀ PMe 3 before and after the cleavage of DNA. Simulations of drug-DNA complexes are now becoming routine to propose atomistic models of DNA-ligand binding and a deeper understanding of the identification, dynamics, and the multiplicity of DNA binding sites.…”

From Interaction to Function in DNA‐Templated Supramolecular Self‐Assemblies

“…These signals were utilized notably to monitor the enzymatic cleavage of DNA. This supramolecular approach allowed the researchers to detect the enzymatic activity in a continuous, label‐free manner . Others reported the use of fluorescence signals of conjugated polymers to detect single‐nucleotide polymorphisms and DNA damages, using dye‐labeled DNA …”